Methylation Confusion and Headaches - (Dec/13/2005 )
I'm seeking a bit of guidance here. I'm at about my limit.
Goal: Using MSP and BSP to view metylation on a promoter region.
700bp region includes the first exon1 of the gene.
Was planning on just pcr'ing Bisulfate treated DNA then Gel purifying and sequencing. I designed and selected primers for both metylated dna and unmetylated dna using MSPprimo.
m5' TAGTTCGTGGTTCGGTCGTGG tm 59
m3' CAAAAAAAAAAAAATCCAACTACGTAA tm 50
u5' TAGTTTGTGGTTTGGTTGTGGG tm 55.8
u3' AAAAAAAAAAAAATCCAACTACATAAAA tm 60
Primers synthed by ITD standard Desalting
DNA was bisulfated treated using ZYmo's DNA methylation GOLD Kit. (heat denaturing)
Samples where then pcr'ed under the following conditions using Roche GC-RICH Taq
PCR machine PE GeneAmp 9600
3min 95 C
30 sec 95 C
30 sec 50 C X 30 Cycles
45 sec 72 C
7 min 72 C
Single postive control was based apon genomic dna, I had concerns about the GC content 75-80% in some regions.
Single postive control on genomic dna yielded correct band ~700bp.
Each MSP primer pair Failed to yield any product.
each primer was test with methblast for alignment. How ever im not sure if the issue is Bisulfate dna quaility (10ul elution), just crappy primer design,bad taq, or the nut trying to make it all work.
Thanks for your time..
You said you are planning to sequence your product, but you are using MSP primers for PCR. To do bisulfite sequencing, primers containing no cpg sites in their sequence should be used. I have never used the program you mentioned but I don't know if the author of the program really understood the biological question.
Another problem with your PCR is the number of cycles which is not enough. For MSP, (the primers you are using) try 35 cycles. For bisulfite sequencing PCR (you need new primers), two rounds of PCR is usually necessary.